The Principal Investigator, Dr. Deplewski, has the long-term goal of pursuing independent investigation in the field of nuclear hormone signaling with emphasis on peroxisome proliferator activated receptor gamma (PPARgamma) signaling. A Mentored Clinical Scientist Development Award will facilitate the growth of Dr. Deplewski's investigative skills and experience by expanding her knowledge of molecular and transgenic techniques through technical training and formal course work, as outlined in the proposed studies. The combination of her learning objectives, the support of her Mentor, Drs. Fredric Wondisford, and the rich research environment at the University of Chicago, will foster Dr. Deplewski's progression to independent lines of investigation into the mechanisms of PPARgamma signaling in the context of adipocyte differentiation and links to insulin action. The Specific Aims of this proposal will focus on the role played by PPARgamma on adipogenesis, with emphasis on the amino terminus which harbors the only coding difference between PPARgamma1 and PPARgamma2, and on the functional differences between the PPARgamma1 and PPARgamma2 isoforms. Three goals are defined: (1) To differentiate the roles of PPARgamma1 and PPARgamma2 in adipocytes by knocking down expression in vitro through the use of RNA interference (RNAI). Several stable clonally derived 3T3-L1 preadipocyte cell lines will be developed using various RNAIs targeting the amino terminus of PPARgamma1 and PPARgamma2. The ability of these lines to undergo differentiation will be studied using cell culture with Oil Red O staining. The expression of PPARgamma target genes will be characterized in these cell lines using microarray analysis and Northern analysis. (2) To perform in vitro mapping of Co-activator binding to the amino terminus of PPARgamma1 verses PPARgamma2, and to determine if a Ser112Ala mutant, which blocks phosphorylation of PPARgamma2, alters co-activator binding and the function of PPARgamma2. (3) To use transgenic techniques to develop mice with a Ser112Ala mutation in PPARgamma2. The degree of obesity and insulin sensitivity will be further studied in these mice.